1. Field of Invention
The present invention relates to a method for optimizing amplification in polymerase chain reaction (PCR), and more particularly, relates to a method for adding element gold, titanium, nickel, bismuth, stibium, selenium, and chromium into polymerase chain reactions to enhance the specificity of the reaction, to increase the yield of target molecules, and to shorten the reaction time.
2. Description of Related Arts
PCR is a quick and easy method for generating a large amount of copies of any DNA target in vitro. Due to the fact that the PCR mechanism is rather complicated, certain interference side effects would be unavoidable in actual practices. For example, there could be mispairing between primers and templates, which cause non-specific amplifications (which is shown as a broad molecular size distribution and non-specific trailing streak) thus resulting to lower amplification specificity and efficiency. Even worse, such side effects could cause the failure of the amplification reaction. The improvement of the PCR amplification specificity is not only determined by the optimizing design of the primer sequence, but also is depended on the optimization of the reaction system and procedure. It is proven that by adding additives, such as formamide, glycerin, DMSO (dimethyl sulfoxide) into the reaction system, the non-specific amplification problems could be ameliorated to certain extent. Unfortunately, the treating effects of above mentioned additives are not ideal in many fields. Moreover, some added components, such as DMSO would inhibit the activities of the polymerase.
U.S. Pat. No. 5,646,019 ‘method for producing primer nucleic acid template’ introduced a method for adding heat-stable single-stranded nucleic acid binding protein (SSB) into the PCR system, wherein SSB protein is only combined with single-stranded DNA, instead of double-stranded DNA. Such method is adapted to inhibit the non-specific amplification. Therefore, the optimization of the PCR amplification could be achieved. However, the techniques adapted for extracting purified SSB are rather complicated. What is more, the reagent purity is required to be higher thus worsening the cost issue. As a result, the reagent kit available in the market would be rather costly. Commonly, the reagent kit prepared with such method will be 6-7 times more expensive than common PCR reagent products. On the other hand, to maintain the biological activities of single-stranded nucleic acid binding protein, the PCR reagent should be reserved at −20° C. temperatures. Finally, the active period of such PCR reagent is rather short.